APPLICATION NOTE Identifiler kits and SeqStudio Genetic Analyzer Authenticating human cell lines using the Identifiler kits and capillary electrophoresis platforms In this application note, we show: The identity of cell lines grown in vitro can be verified using the Applied Biosystems Identifiler Plus and Direct kits Cell lines can be authenticated using as little as 100 pg of purified genomic DNA (gdna) Cell lines can be authenticated directly from punches of cells spotted onto Applied Biosystems NUCLEIC- CARD sample collection devices The new Applied Biosystems SeqStudio Genetic Analyzer gives higher performance with lower concentrations of gdna and cells than Applied Biosystems 3130 and 3500 Genetic Analyzers Introduction The study of human diseases relies heavily on the analysis of dissociated human cell lines grown in culture. However, an increasingly acknowledged problem is that cells grown in vitro can be misidentified or become contaminated with other unrelated cell lines [1]. Misidentification of cell lines produces misleading results, confusion, and added costs to research [2-4]. Journals and funding agencies now require researchers to ascertain that the cell lines they have used are authentic, and to identify strategies for ensuring they remain so over the course of a study (for examples, see Yu et al., 2015 [5] and Neimark, 2015 [6]). Establishing the authenticity or provenance of a cell line often involves analysis of variants or alleles of several loci and making sure these variants match expected alleles. There are several methods for analyzing variant loci, ranging from electrophoretic analysis of isozyme variants through analysis of restriction fragment length polymorphisms (RFLPs) and amplified fragment length polymorphisms (AFLPs), to next-generation sequencing (NGS) and analysis with MALDI-TOF mass spectrometry. However, many of these methods suffer from at least one drawback, ranging from insufficient complexity to high costs. Nevertheless, analysis of highly variable short tandem repeat (STR) markers, a well-established technique commonly used in DNA forensic analysis, can provide a simple, inexpensive, and highly specific genetic fingerprint of a cell line. Comparing a profile of alleles present at these highly variant loci to known, standardized samples of a cell line provides confidence that the cell line is authentic. Organizations such as ATCC and Leibniz-Institute DSMZ German Collection of Microorganisms and Cell Cultures provide online access to searchable databases that allow investigators to query known cell types. Alternatively, researchers can establish an allelic profile of cell lines unique to their lab, and over time compare the allelic profiles of the cells to their own internal standards to ensure that the cell identities are true.
Typically, analysis of STRs is performed by capillary electrophoresis (CE) of fragments amplified from microsatellite loci with varying number of repeats. We have established the gold standard for CE instrumentation by developing and offering instruments that are optimized for researchers needs in sensitivity and throughput. Furthermore, the Applied Biosystems product portfolio has several different kits for PCR-based STR fingerprinting for use on CE instruments. The Applied Biosystems Identifiler Plus PCR Amplification Kit has been optimized to analyze 16 highly variant human STRs over a wide range of purified gdna preparations. The Applied Biosystems Identifiler Direct PCR Amplification Kit was first developed to analyze the same 16 STR loci, starting from dried blood or buccal spots (for example, on NUCLEIC-CARD devices) or buccal swabs. For the NUCLEIC-CARD device, a 1.2 mm punch from the card is placed directly into a PCR tube or well, and amplified without any further purification. Finally, Applied Biosystems GeneMapper Software 5 facilitates analysis of STRs by making use of pre-established allelic ladders and sizing bin sets for the various STR alleles covered by the Identifiler kits. An illustration of the complete workflows for cell line authentication is shown in Figure 1. We have recently introduced a new member of the CE instrument family the SeqStudio Genetic Analyzer with several new features aimed at making CE analysis easier. The SeqStudio Genetic Analyzer has a completely redesigned user interface driven by an on-instrument touch screen and integrated computer, facilitating setup and runs. Embedded run modules and a removable slide-in cartridge allow the SeqStudio Genetic Analyzer to be used for either Sanger sequencing or fragment analysis without any reconfiguration. This genetic analyzer provides maximum flexibility and ease of use, making it an ideal platform for many applications, such as cell line authentication, when coupled with the Identifiler kits. A Spot cells onto NUCLEIC-CARD device Amplify using Identifiler Direct kit Fragment analysis by CE Analyze using GeneMapper Software 5 B Purify gdna Amplify using Identifiler Plus kit Fragment analysis by CE Analyze using GeneMapper Software 5 Figure 1. Workflows for cell line authentication. Two methods are available for cell line authentication. (A) Cells can be spotted onto NUCLEIC-CARD devices, punches of the cards amplified directly using the Identifiler Direct kit, and fragments analyzed on Applied Biosystems CE instruments using GeneMapper Software 5. (B) Alternatively, gdna can be purified from cell lines, amplified using the Identifiler Plus kit, and fragments analyzed by capillary electrophoresis and GeneMapper Software 5.
Results Use of Identifiler Plus kit and gdna GeneMapper Software 5 simplifies the calling of STR alleles by aligning unknown fragments with a ladder of STR fragments of known allele sizes (Figure 2, top). For example, the sample containing gdna from cells has two peaks from the sample at the D7S820 locus. GeneMapper Software 5 aligns these two peaks to alleles 8 and 10 in the allelic ladder, with sizes of approximately 263 and 271 bp, respectively, without any other significant peak being present (Figure 2, bottom). Thus, at this locus, the cells are heterozygous for these two alleles. At the D19S433 locus, a single peak is present at allele 14, indicating that these cells are homozygous for this allele. The combination of all the alleles gives a unique fingerprint to this cell culture, and can be compared to other cultures or known samples to establish authenticity of this culture. Figure 2. GeneMapper Software 5 analysis that compares the Applied Biosystems Identifiler Plus Allelic Ladder (top) and purified gdna from cells grown in culture (bottom). GeneMapper Software 5 uses the allelic ladder provided in the Identifiler kits to assign the alleles present in an unknown sample to known STR alleles. The boxes below the peaks show the allele number, the height of the peak, and the size of the fragment in base pairs.
To show the utility of the Identifiler Plus kit and SeqStudio instrument for cell line authentication, we analyzed DNA from cultures of five different commonly used cell lines. Briefly, gdna was purified from cell pellets using the Invitrogen RecoverAll Total Nucleic Acid Isolation Kit for FFPE. However, since deparaffinization was not necessary for this sample type, we skipped those steps in the protocol. The DNA amount was determined using the Applied Biosystems Quantifiler Human DNA Quantification Kit. One nanogram of purified DNA in 10 µl water, or 3-fold serial dilutions of gdna starting at 3 ng/µl in water, were prepared and analyzed with the Identifiler Plus kit according to the protocol found in the User Guide (Pub. No. 4440211, Revision F). Following PCR, 1 µl of the reaction was denatured in Applied Biosystems Hi-Di Formamide, and loaded onto the instrument for capillary electrophoresis. Fragment peaks were analyzed in GeneMapper Software 5 using an imported human identification (HID) analysis method (see Appendix). Initial experiments were performed to determine the number of PCR cycles needed for optimal allele detection on the different instrument platforms (Figure 3). Accuracy was determined by comparing and confirming with the alleles present in the ATCC database (see later section, Verifying cell authenticity against known standards) for these five cell lines. On the 3130 instruments, 27 29 cycles generated the highest percentage of accurate calls, whereas on the SeqStudio and 3500 instruments, 27 28 cycles produced highest-confidence data. Using these results as a guide, we recommend determining the optimal number of cycles for the amount of DNA and CE system that will be used in your lab. To determine the minimal amount of DNA that can be used with the Identifiler Plus kit, we also performed serial dilutions of purified gdna (data not shown). The most accurate results were obtained when 0.3 3 ng of gdna was used in the amplification reaction. Note that by using the maximal number of PCR cycles, we were able to accurately profile small amounts of DNA; however, at higher DNA concentrations, more PCR cycles also resulted in higher numbers of spurious peaks and reduced accuracy. Therefore, for highest confidence in the allelic calls, we recommend using 1 ng of purified gdna in a cell line authenticity analysis. Correct calls 100% 90% 80% 70% 60% 50% 40% 30% SeqStudio Genetic Analyzer 3500 Genetic Analyzer 3130 Genetic Analyzer 20% 10% 0% 26 cycles 27 cycles 28 cycles 29 cycles Number of PCR cycles Figure 3. Analysis of genomic DNA using the Identifiler Plus kit. One nanogram of DNA from five different cell lines was analyzed using varying numbers of PCR cycles. Overall, the most accurate results were obtained when PCR was performed for 27 29 cycles.
Use of Identifiler Direct kit and NUCLEIC-CARD device An alternative method for authenticating cell cultures makes use of the NUCLEIC-CARD device. The matrix in these cards is chemically treated to enable cell lysis and protein denaturation so that the DNA on the card is immobilized and preserved for long-term storage at room temperature. To show performance using these cards, we prepared suspensions of several different human cell lines in PBS (approximately 5 x 10 5 cells/ml; see Figure 4 for exact concentrations). One hundred microliters of suspension were spotted directly onto the NUCLEIC- CARD device and dried overnight. Single 1.2 mm punches were taken from the area with the dried suspension and placed into a well of a 96-well PCR plate. Controls and reagents from the Identifiler Direct kit were added to the plate according to the protocol supplied in the user guide (Pub. No. 4415125, Revision J), and amplified for 29 cycles. As described above, following PCR amplification, 1 µl of product was denatured in Hi-Di Formamide and analyzed on a SeqStudio, 3500, or 3130 Genetic Analyzer using GeneMapper Software 5. The minimum amount of cell suspension that can be analyzed was determined by performing serial dilutions of the starting cell suspension into PBS (Figure 4). Complete and accurate profiles were obtained in the undiluted samples across all cell lines. Diluting the suspension 10-fold before spotting onto the NUCLEIC-CARD device resulted in dropout of some alleles. The number of allele dropouts correlated with the starting concentration of the suspensions those with slightly higher concentration had fewer dropouts, while lower concentrations had more dropouts. At a 100-fold dilution, an average of about 50% of the alleles were detectable. As before, suspensions with slightly higher starting concentration had less dropout. For highest confidence in allele calls, we therefore recommend starting by spotting a suspension of around 5 x 10 5 cells/ml onto the NUCLEIC-CARD device. Cell line 2.6 x 10 5 4.7 x 10 5 U2OS 7.2 x 10 5 2.1 x 10 5 HEK 5.0 x 10 5 Correct calls 100% 80% 60% 40% 20% 0% SeqStudio Genetic Analyzer 3500 Genetic Analyzer 3130 Genetic Analyzer Starting concentration (cells/ml) Undiluted 10-fold 100-fold Figure 4. Titration of cell suspensions dried onto NUCLEIC-CARD device. Cell suspensions were prepared at the concentrations shown in the table, then serial dilutions of those suspensions were analyzed using NUCLEIC-CARD devices and the Identifiler Direct kit. The average percentages of correct allele calls across all cell lines at the indicated dilutions are shown.
Verifying cell authenticity against known standards Several research organizations have recognized the need for matching unknown samples against known cell lines to establish authenticity. For example, ATCC has set up a web-based query against their database of cell line STRs (Figure 5, atcc.org/str_database.aspx). To use it, simply enter the alleles in the test sample and choose the stringency of the query. A list of cell lines in the ATCC database that match the STRs present in the test sample will be returned. Similarly, the Leibniz-Institute DMSZ has a web-based STR query system in place (dsmz.de/fp/ cgi-bin/str.html). Note that both of these pages query only 9 loci (8 autosomal and 1 sex-linked), and therefore not all of the loci in the Identifiler kits are used for this external database comparison. However, these additional loci are enormously useful for comparisons against locally generated controls or when establishing the provenance of a new cell line. The identity of each of the cell lines described in this application note was verified using both databases. These organizations have therefore made it possible to compare alleles present in an unknown sample to those of known, commonly used cell lines, simplifying confirmation that a cell line is genuine. The International Cell Line Authentication Committee has set guidelines for interpretation, that cells with 80 100% allelic matching come from the same donor, and a <50% match generally means that cells come from different donors or have different origins [7]. Identification of contaminating cells in a culture One of the objectives of a cell line authentication solution is to determine whether a cell line is contaminated with unrelated cells. This is easily achievable, since the contaminating cells are likely to have a different STR profile than the parent line. In a mixture of cell lines, the final profile will reflect the combination of the genomic makeup of all cells present. For example, a single peak at a locus could represent both cell types being homozygous for the same allele, or one cell type being homozygous for the allele and the other homozygous for a deletion that removes the locus. Two peaks could reflect both cell types being homozygous for different loci, heterozygous for the same loci, etc. Although the interpretation of aberrant peaks at a single locus could be challenging and ambiguous, analysis of 16 different loci will likely identify distinct peaks that clearly point to the presence of a contaminating cell line, even if the genomic makeup of the contaminating cell line might not be fully discernible. To test the limit of detection of contaminating cells in a culture, we prepared cell suspensions of 5 x 10 5 cells from cells and cultures, then mixed the suspensions such that they contained 10%, 15%, 20%, 25%, 30%, and 50% cells; 100 µl of each mixed cell suspension was spotted onto the NUCLEIC-CARD device. Card punches (1.2 mm) were obtained once the suspensions were dried, amplified with Identifiler Direct reagents for 29 PCR cycles, and analyzed on the three different instrument platforms. Similarly, we mixed gdna with gdna at 1%, 2%, 3%, 4%, 5%, and 10% proportions. One nanogram of each mixture was amplified using the Identifiler Plus kit and 29 PCR cycles, and analyzed on the SeqStudio Genetic Analyzer. Figure 5. Cell line identities can be verified by uploading allele calls to the ATCC cell line STR database. Once the alleles for each locus are entered, cell lines that match the STR profile are shown. Note that the ATCC database requires queries for 8 loci, all of which are covered in the Identifiler kits. The database can be accessed at atcc.org/ STR_Database.aspx. For details, see the tutorial on the ATCC STR database page.
An equal mixture of and cells produces a profile in which extra peaks are clearly present (Figure 6A). For example, at the locus D7S820, three alleles were positively located, whereas two alleles, at most, would be expected in a homogeneous diploid cell population. Several other loci also have three alleles present. Although conclusions can t be drawn solely from the one- and two-peak loci, the presence of three alleles at multiple loci definitely points to a heterogeneous cell population or a DNA mixture. On all platforms, we could clearly detect heterogeneity in the mixture containing 20% cells (Figure 6B). As the percentage of cells dropped in the mixture, the number of -specific alleles detected also decreased. However, the decrease was less marked A on the SeqStudio and 3500 platforms even at 10% of the cell mixture, about 65% of the -specific alleles were still detectable. A similar analysis was performed using lower percentages of gdna from cells mixed with DNA (Figure 6C). Over half of the -specific alleles could be detected in mixtures containing as little as 4% cells, demonstrating the sensitivity of the Identifiler Plus kit. Contaminating cells with nearly complete profiles (highest confidence) can therefore be detected in a population if they are present at greater than 20% of the total population. However, indicator alleles may be detectable if the population contains as little as 4% of a contaminating cell line. B Unique allele detected 100% 80% 60% 40% 20% 0% SeqStudio Genetic Analyzer 3500 Genetic Analyzer 3130 Genetic Analyzer 10% 15% 20% 25% 30% 50% cells in population C Unique allele detected 80% 70% 60% 50% 40% 30% 20% 10% 0% 1% 2% 3% 4% 5% 10% gdna Figure 6. Detection of contaminating cells in a culture. (A) Extraneous peaks in a culture may be an indication of genomic heterogeneity, or the presence of contaminating cells. In this case, a 1:1 mixed suspension of and cells was analyzed using the NUCLEIC-CARD device and Identifiler Direct kit. The profile of a contaminated culture can vary, depending on the allelic makeup of the host and contaminating cells. (B) Analysis of mixed samples on the NUCLEIC-CARD device. cells and cell suspensions were diluted to 5 x 10 5 cells/ml each, mixed in the indicated proportions, and spotted onto the NUCLEIC-CARD device. Identical aliquots were analyzed on the indicated instrument types. Note that the 30% population was not analyzed on the 3500 platform. (C) Analysis of gdna from mixed samples. gdna from cells was mixed with gdna from cells at the indicated proportions. More than half of the alleles unique to cells can be detected in a mixture containing at least 4% gdna.
Allelic imbalance in cell lines In analyses using Identifiler kits, genotypes present themselves as either a single peak (homozygous) or two balanced peaks with equal height (heterozygous). However, cell lines passaged in vitro demonstrate genomic instability, and the peak heights of sister alleles can differ due to duplication or deletion of loci, portions of chromosomes, or entire chromosomes. For example, the HEK cells used in this study are balanced at the D8S1179 locus, but show significantly different peak heights at the CSF1P0 and D13S317 loci (Figure 7). Moreover, when the DNA quantity added to the amplification reaction is 100 pg or less, stochastic effects may introduce imbalances in peak height. To demonstrate that the Identifiler kits are detecting true allelic imbalances, we analyzed 8 different standardized gdna preparations (1 ng) from normal human donors. We determined the allelic balance by calculating the ratio of peak heights of alleles detected at each locus. For heterozygous loci, this ratio is 1.0; for homozygous loci we defined the ratio as 1.0 for this study. We simultaneously analyzed 1 ng of gdna from the cell lines described above. Most of the loci from the standardized human gdna showed allelic balance, with ratios very close to 1.0 when 1 ng of DNA was used (Figure 8). There were a few exceptions two individual donors had a locus with a ratio of 1.5, suggesting they might contain a duplication of an allele (3:2), and one individual had a manifold duplication of the TH01 allele locus. However, the cell lines showed a vast diversity of allele ratios, ranging from 0.2 to 4.5. Since the kit was able to accurately measure normal allele ratios, this potentially reflects genomic instability of the cell lines through passaging over the years. Conversely, cells that have not been passaged over long periods, such as primary cells or uninduced stem cells, are not likely to demonstrate the same allelic imbalance. Figure 7. Allelic imbalance in cell lines. In normal cells, the peak height of heterozygotes is very similar; in cell lines, peak heights can often differ at a locus (red circles). This is possibly due to genomic instability of the cell line, leading to duplication and deletions of loci and therefore different abundances of alleles. 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 AMEL CSF1PO D13S317 D16S539 D18S51 D19S433 D21S11 D2S1338 D3S1358 D5S818 D7S820 D8S1179 FGA TH01 TPOX vwa Figure 8. Cell lines demonstrate measurable allelic imbalance. Eight standardized gdna samples from human donors (left of the green line) and five cell lines (right of the green line) were analyzed with the Identifiler Plus kit. Allele ratios were calculated using the heights of peaks in heterozygotes, or defined as 1.0 in homozygotes (red line). Note that most of the samples from normal human donors have allele ratios close to 1.0, whereas the cell line samples often have deviations from unity.
Conclusions In this application note, we have described simple and inexpensive workflows for cell line authentication. We demonstrated that the Identifiler Plus and Identifiler Direct kits can be used to distinguish STR allelic fingerprints in in vitro cultured cells. In fact, because the Identifiler kits query 16 STR loci on 15 different chromosomes, they provide exceptional flexibility for detecting changes in ploidy in cell lines, when querying DNA from degraded samples, and in future analyses as cell line derivatives and databases become more numerous and complex. We showed that results can be obtained from as little as 0.1 ng of gdna. However, to ensure optimal results with highest confidence, we recommend 1 ng of gdna. We also showed that authentication results could be obtained from 100 µl of a suspension of 5 x 10 5 cells/ml immobilized on a NUCLEIC- CARD device. We demonstrated that our capillary electrophoresis instruments perform equivalently when gdna or cell concentrations are relatively high. Finally, we showed that the SeqStudio Genetic Analyzer gives higher performance with lower concentrations of gdna and cells compared to the 3130 and 3500 Genetic Analyzers. Notably, the initial sample preparation, PCR amplification, preparation for CE, and downstream analysis methods are nearly identical across the entire CE instrument line. This provides an opportunity for researchers to choose the platform most appropriate for their laboratory s throughput and budget. References 1. Lorsch JR et al. (2016) Fixing problems with cell lines. Science 6216:1452 1453. 2. Huang Y et al. (2017) Investigation of cross-contamination and misidentification of 278 widely used tumor cell lines. PLoS One 12(1):e0170384. doi: 10.1371/journal.pone.0170384. 3. He Y et al. (2016) Retracted: Knockdown of tumor protein D52-like 2 induces cell growth inhibition and apoptosis in oral squamous cell carcinoma. Cell Biol Int 40:361. 4. https://grants.nih.gov/grants/guide/notice-files/not-od-08-017.html 5. Yu M et al. (2015) A resource for cell line authentication, annotation and quality control. Nature 520:307 311. 6. Neimark J (2015) Line of attack. Science 347: 938 940. 7. http://standards.atcc.org/kwspub/home/the_international_cell_line_authentication_ committee-iclac_/authentication_sop.pdf
Appendix GeneMapper modules and settings needed for analysis Before analyzing the FSA files for a cell line authentication project, the appropriate BIN files must be imported into GeneMapper Software 5. To import the files needed, follow the instructions below. AmpFlSTR Bins_v2.txt and AmpFLSTR_Panels_v2.txt can be downloaded here: thermofisher.com/us/en/ home/technical-resources/software-downloads/ genemapper-id-software.html Launch GeneMapper Software 5. Select Tools from the menu bar. To install the Panel File : Single-click on Panel Manager in the left-hand window pane. Select File and import Panels from the menu bar. A dialog box will appear. Navigate to the location of the Panel file on your computer. If you placed it in the default Panels folder, the dialog box should open to the correct folder. Select the file titled AmpFlSTR_Panels_v2.txt This will install the Panel in the left window pane. Select Panel Manager. Run modules for each instrument used in this study. Instrument Polymer used Injection voltage (kv) Injection time (sec) Run voltage (kv) Run time (sec) Capillary length (cm) Run module 3130xl POP-7 1.2 23 15.0 1,200 36 cm FragmentAnalysis36_POP7 3500xL POP-7 1.6 15 19.5 1,330 50 cm FragmentAnalysis50_POP7xl SeqStudio POP-1 2.0 10 9.0 1,440 28 cm FragAnalysis
To install the Bin File : Single-click on AmpFlSTR_Panels_v2 in the left-hand window pane. Select File and import Bin Set from the menu bar. A dialog box will appear. Navigate to the location of the Bin file on your computer. If you placed it in the default Panels folder, the dialog box should open to the correct folder. Select the file titled AmpFlSTR_Bins_v2.txt This will install the Bins in the pull-down menu at the top titled Bin Set. Click on Apply, followed by OK to return to GeneMapper Software 5. The Identifiler_v2 panel and associated Bin Set can be chosen under Panel when performing analysis. To set up a cell line authentication analysis method, either modify an existing method or create a new one using the values in Figure 9A as a guide. To set up a plot setting to visualize the alleles detected, create a new setting and enter the parameters shown in Figure 9B. To view more than two alleles in the genotyping tables, change the Allele Settings in the Genotypes tab of the Table Setting Editor (Figure 9C). A B C Figure 9. GeneMapper Software 5 settings for (A) cell line authentication analysis method, (B) cell line authentication plot settings, and (C) viewing more than two alleles in the genotyping tables. These screens can be accessed by opening the Manager Tool and choosing the appropriate tab.
Ordering information Product Quantity Cat. No. SeqStudio Genetic Analyzer 1 instrument A34274 SeqStudio Analysis Software 1 each 44443764 SeqStudio Cartridge 500 reactions A33671 SeqStudio Starter Kit 1 kit A35000 SeqStudio Full-Day SmartStart Training 1 each A34684 3500 Genetic Analyzer 1 system 4440466 3500xL Genetic Analyzer 1 system 4440467 Identifiler Plus PCR Amplification Kit 100 reactions A26364 Identifiler Direct PCR Amplification Kit 200 reactions 4467831 NUCLEIC-CARD matrix, 1 spot 100 cards 4473973 NUCLEIC-CARD COLOR matrix, 4 spots 50 cards 4473978 RecoverAll Total Nucleic Acid Isolation Kit for FFPE 40 reactions AM1975 GeneMapper Software 5 1 license 4475074 Learn more about the SeqStudio Genetic Analyzer at thermofisher.com/seqstudio For Research Use Only. Not for use in diagnostic procedures. Identifiler kits and Identifiler Direct kits cannot be sold in China or Japan for purposes outside of Forensics or Paternity until November 29, 2019. 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. NUCLEIC-CARD is a trademark of Copan Flock Technologies. COL04325 1017